Growth, Purification, and Analysis of GBR22 Protein
Introduction Use of recombinant proteins is now a common sight in scientific research. With numerous methods to produce a recombinant protein, protocols to express, purify, and characterize a protein must be decided for each individual protein.
E. coli serves as an outstanding expression host for the recombinant production of a wide variety of proteins due to its convenience and low cost. [1] More specifically, BL21(DE3) is an excellent host strain with deficiency in both lon and ompT proteases, which can enhance accumulation by reducing proteolytic degradation. [2]
In order to purify the protein, bacterial lysate is a critical step to maximize the fraction of the extracted recombinant protein. Although mechanical lysis can be performed through homogenization or sonication, this lab used lysozyme to degrade the cell-wall material and Benzonase to digest the DNA and RNA materials [2]. By adding intermediate concentration of imidazole, weakly bound contaminants can be filtered while retaining the recombinant proteins.
In order to characterize the purified protein, both SDS- PAGE analysis and UV absorption spectroscopy can be used. After staining with a dye, the intensity of the bands relatively represents the amount of the protein. Moreover, SDS-PAGE allows the purity of the sample to be estimated. Lastly, the UV-Vis spectroscopy can be used to estimate the concentration of the purified protein. [1]
The objective of the lab was to express and analyze purified gbr22 protein initially grown in bacteria using gel electrophoresis and UV- Vis spectroscopy. After lysing the cell, centrifuging, and using Ni-NTA affinity purification, the gbr22 protein should only remain in the sample.
Methods & Materials For the protein expression, there were two transformation tubes each containing 25ul competent bacterial cells (New England Biolab, Ipswich, MA), but the experimental plate with DNA contained extra 1-2ul of plasmid. After heat shocking the tubes in 42°C water bath for 45 seconds and adding 200ul of SOC media, the bacteria/SOC mixtures for the control and DNA were transferred and evenly spread onto each plate and grown overnight in 37°C.
The starter culture was grown by adding a single bacterial colony from the LB/agar plate to each tube containing 5mL of LB supplemented with 10mL ampicillin. The tubes with the sterile tips were placed in the shaking incubator (37°C at 200-350 rpm) and grown for 8 hours. Then, a larger culture was grown by transferring 0.625mL of the starter culture in fresh 25mL LB and 50ul of ampicillin in a 125mL Erlenmeyer flask. The flask covered with foil was placed in the shaking incubator to be grown for 16-24 hours. After the media turned purple, the larger benchtop centrifuge was used to harvest the cell. After spinning down the cell, sample 1 was taken and stored in 4°C (same location that the later samples were stored). Finally, the bacteria was transferred into a 50mL conical tube to be centrifuged for 10 minutes at 5,000 rpm at 4°C. After decanting to save the purple pellet, 2.5mL of 1x PBS was added. Then, 60ul of lysozyme was added to the vortexed tube. The tube was stored at -20°C.
After thawing the conical tube, 2ul of Benozase were added and incubated at room temperature. The lystate was distributed into several 1.7mL microcentrifuge tube and the tubes were centrifuged for 20 minutes at 14,000rpm at 4°C. 50ul of the supernatant was taken and labeled as sample 2. The liquid supernatant was transferred into a 15mL conical tube leavening the insoluble materials behind.
The lysate was filtered through a syringe filter with a 5 mL syringe into a 14mL round bottom transformation tube. Then, 0.5mL of Ni-NTA resin/buffer mix was added. The apparatus shown to the right was set up along with an ice bucket and column (Bio Rad Laboratories, Hercules, CA). After running nanopure water andresin/buffer through the column into the 10mL “waste” round bottom tube, 50ul was taken from the waste solution and was labeled as sample 3. The Ni-NTA resin was washed with 5ml of 20mM imidazole in 1XPBS. The solution was collected into the 10ml “wash” round bottom tube. 50ul was sampled and were labeled as sample 4.
After adding 50mL of the buffer containing 250 mM of imidazole, the buffer was collected in a 15mL conical tube labeled as “Elution 1”. Next, 5 mL was elution buffer was added again and the solution was collected in a 15mL conical tube labeled as “Elution 2”. In the end, 50ul of each elution1 and elution 2 was taken and they were labeled as sample 5 and sample 6.
By using the Nanodrop spectrophotometer (thermo Scientific, Wilmington, DE) with elution #1, the concentration of the purified protein was estimated. The absorptions were measured at both Protein A280 mode and UV/VIS mode. After searching the extinction coefficient online along with the absorbance measured, both the concentration and yield of the purified protein were measured at 280 nm and maximal wavelength.
In order to prepare the SDS-PAGE gel samples, sample 1 was centrifuged at 5 minutes at 5,000 rpm. After decanting the liquid, 200ul of water and 40ul of loading buffer was added. For samples 2-6 (4-6 from partner), 10ul of 6x loading buffer was added. All the samples were placed in a heat block at 95°C for five minutes and were later centrifuged for 2minutes at 5,000 rpm. After assembling mini-PROTEAN tank (Bio Rad Laboratories, Hercules, CA), each lane was cleaned using needle and syringe. In the first lane, 7ul of MW standard (Fermentas, Glen Burnie, MD) was added. Afterwards, 20ul of each protein samples was sequentially added (MW-1-2-3-4-5-6-[4-5-6]). The gel ran for 25 minutes at 200V. After sufficiently rinsing the gel, ~30mL of imperial protein stain was added. The container with the gel was placed on the orbital shaker for 1.5 hours. After rinsing the gel twice, KimWipe was placed in the container and the gel was washed overnight. After placing the gel on a Whatman filter paper and covering with saran wrap, the gel was dried at 75°C on gradient cycle for 1.5 hours.
Results:
Figure: Starter culture containing BL21 (DE3) and Amp (Experimental plate containing pGEM-gbr22 shown on the left & Control plate with no DNA control shown on the right)
Figure 2: Large culture of BL31(DE3) & pGEM-gbr22 grown in LB and Amp for 24 hours in a shaking incubator at 37 degrees Celsius and 200-350 rpm
Figure 3: Cell pellet grown from large culture after being centrifuged for 10 minutes at 5,000 rpm at 4 degrees Celsius
Wet Pellet Weight: .42g
Figure 4: Elution #1 (Left) and Elution #2 (Right) containing purified pGEM-gbr22 protein
Most gbr22 protein contained in Elution #1 after lysing cell, centrifuging, and filtering through Ni-NTA affinity purfication Elution #2 as backup
Figure 5: Using Nanodrop spectrophotometer on Protein A280 setting with Elution #1 containing purified gbr22 protein Trial #1
Absorption at 0.896 at wavelength of 280nm
Figure 6: Using Nanodrop spectrophotometer on Protein A280 setting with Elution #1 containing purified gbr22 protein Trial 2
Absorption at 0.869 at wavelength of 280nm
Figure 7: Using Nanodrop spectrophotometer on UV-Vis Mode with Elution #1 containing purified gbr22 protein Trial #1
Absorption measured at 0.136 at the wavelength of 574 nm
Figure 8: Using Nanodrop spectrophotometer on UV-Vis Mode with Elution #1 containing purified gbr22 protein Trial #2
Absorption measured at 0.126 at the wavelength of 574 nm Beer's Law Calculation
>Determining Concentration of Protein using 280nm
Molecular weight: 25794.2 g/mol
Extinction Coefficient: 38850 M^-1*CM^-1
A=Ebc
A=(.896+.869)/2 =.8825
.8825= c (1) (38850 M^-1*CM^-1)
c=.00002273 mol/L
>Determining Concentration of Protein using Maximal Wavelength
A= (.136+.126)/2 = .131
.131 = c (1) (118,300 M^-1*CM^-1) c= .00000111 mol/L Yields from Nanodrop Spectrophotometry
>280nm wavelength: .00002775 mg
(.0002272mol/L)(25794.2g/mol)= .00000555 g/L
.00000555 mg/ml (5ml)= .00002775 mg
>Maximal wavelength: .057926312 mg
(.00000111mol/L)(25794.2g/mol)= .02863156 g/L
(.0286315mg/mL) (5mL) = .05792312 mg
Figure 9: Result of Gel Electrophoresis Using mini-PROTEAN of Various Samples taken during Purification Step for gbr22
(Left to Right: MW-1-2-3-4-5-6-[4-5-6 sample from partner])
Figure 10:Image of Ladder for PageRuler/ Fermentas of MW (kDa) for reference
Discussion Since the lab outlined a detailed procedure, there could be numerous sources of error that could mislead the data. When working with E. coli and bacterial media in protein expression, aseptic technique was crucial to prevent contamination. For example, a gas burner was turned on to sterilize the general area when handling the bacteria. If the tubes were left open for a long period of time or proper sterile equipment was not used, the results would show inconsistencies. In addition, including bacteria, that are not resistant to ampicillin or contain mutation, would provide inaccurate outcome from protein purification and characterization. Lastly, the lab required using unfamiliar equipment, such as the Nanodrop spectrophotometer and mini-PROTEAN tank. Misusing the equipment could lead to incorrect reading of measurements that could significantly impact the results.
The protein purification steps successfully separated the protein from the overexpressed bacteria. By adding lysozyme and Benzonase, the soluble materials held within the cell were released. Then, the centrifugation removed the insoluble large cell materials. Using affinity tag and Ni-NTA resin, all proteins except gbr22 were filtered.
Samples 1-6, used for gel electrophoresis, represented various specimens taken during the protein expression and purification steps. The first sample taken contains the gbr22 protein, which includes both soluble and insoluble cell materials grown overnight before any filtration. Before the second sample, lysozyme was used to break down the cell wall and lyse the cell. Moreover, Benzonase digested the DNA/RNA and reduced the viscosity of the mixture. As a result, sample 2 contained everything except the large insoluble protein particles filtered by centrifugation. Sample 3 involved a simple flow, which kept all the proteins including gbr22. Before taking the fourth sample, a small amount of imidazole was added to remove the proteins that were only loosely bound to the resin. Sample 5 contained the purified gbr22 proteins detached from the nickel beads since a large amount of imidazole was added. Lastly, sample 6 contained the remaining purified proteins that were not released in sample 5. The significant changes in the amount of cellular materials in each sample can be seen in Figure 9.
Wash buffer contained less concentrated imidazole that washed away other proteins with two or three histidines while keeping the gbr22 protein with six histidines. In comparison, the elution buffer contained concentrated imidazole that had the ability to push out the gbr22 protein. These two buffers were used in the experiment since the gbr22 protein has been modified to have six histidine residues attached to the C-terminus. As a result, the histidine residues were attached to the nickel beads in the column. Adding a large amount of imidazole competes with the histidine residues for metal binding. As a result, the gbr22 protein was finally be released.
Then, the elution #1 shown in Figure 4 was used for Nanodrop spectrophotometer in order to find the absorbance at 280nm and maximal wavelength. After obtaining the extinction coefficient from external sources, the concentrations of the protein were calculated through Beer’s Law. Moreover, the amount of purified protein in mg was calculated at 280nm and maximal wavelength by knowing volume and concentration of the purified protein.
Using the dried gel and protein ladder of Molecular Weight standard in Figure 10, the purified protein was estimated to be 28 kDa. As shown in samples 2-4 in Figure 9, the presence of an extra band represented contamination. However, multiple filtrations between the samples decreased the intensity of the extra band with the progression of the lab. In the end, sample 5 contained closely purified gbr22 protein.
Conclusions The purpose of the lab was to overexpress a recombinant protein in E. coli, to purify gbr22 protein through various filtration methods, and to analyze the samples through gel electrophoresis and Nanodrop spectrophotometer. Verified through the gel, the gbr22 protein was closely purified with little interference from extra cellular materials in sample 5. The techniques gained throughout the lab would frequently be used in independent research when a specific protein needs to be isolated to bind with a top ranking ligands from GOLD.
References [1] Nat Methods. Protein production and purification. 2008, 5(2):135-46.
Introduction
Use of recombinant proteins is now a common sight in scientific research. With numerous methods to produce a recombinant protein, protocols to express, purify, and characterize a protein must be decided for each individual protein.
E. coli serves as an outstanding expression host for the recombinant production of a wide variety of proteins due to its convenience and low cost. [1] More specifically, BL21(DE3) is an excellent host strain with deficiency in both lon and ompT proteases, which can enhance accumulation by reducing proteolytic degradation. [2]
In order to purify the protein, bacterial lysate is a critical step to maximize the fraction of the extracted recombinant protein. Although mechanical lysis can be performed through homogenization or sonication, this lab used lysozyme to degrade the cell-wall material and Benzonase to digest the DNA and RNA materials [2]. By adding intermediate concentration of imidazole, weakly bound contaminants can be filtered while retaining the recombinant proteins.
In order to characterize the purified protein, both SDS- PAGE analysis and UV absorption spectroscopy can be used. After staining with a dye, the intensity of the bands relatively represents the amount of the protein. Moreover, SDS-PAGE allows the purity of the sample to be estimated. Lastly, the UV-Vis spectroscopy can be used to estimate the concentration of the purified protein. [1]
The objective of the lab was to express and analyze purified gbr22 protein initially grown in bacteria using gel electrophoresis and UV- Vis spectroscopy. After lysing the cell, centrifuging, and using Ni-NTA affinity purification, the gbr22 protein should only remain in the sample.
Methods & Materials
For the protein expression, there were two transformation tubes each containing 25ul competent bacterial cells (New England Biolab, Ipswich, MA), but the experimental plate with DNA contained extra 1-2ul of plasmid. After heat shocking the tubes in 42°C water bath for 45 seconds and adding 200ul of SOC media, the bacteria/SOC mixtures for the control and DNA were transferred and evenly spread onto each plate and grown overnight in 37°C.
The starter culture was grown by adding a single bacterial colony from the LB/agar plate to each tube containing 5mL of LB supplemented with 10mL ampicillin. The tubes with the sterile tips were placed in the shaking incubator (37°C at 200-350 rpm) and grown for 8 hours. Then, a larger culture was grown by transferring 0.625mL of the starter culture in fresh 25mL LB and 50ul of ampicillin in a 125mL Erlenmeyer flask. The flask covered with foil was placed in the shaking incubator to be grown for 16-24 hours. After the media turned purple, the larger benchtop centrifuge was used to harvest the cell. After spinning down the cell, sample 1 was taken and stored in 4°C (same location that the later samples were stored). Finally, the bacteria was transferred into a 50mL conical tube to be centrifuged for 10 minutes at 5,000 rpm at 4°C. After decanting to save the purple pellet, 2.5mL of 1x PBS was added. Then, 60ul of lysozyme was added to the vortexed tube. The tube was stored at -20°C.
After thawing the conical tube, 2ul of Benozase were added and incubated at room temperature. The lystate was distributed into several 1.7mL microcentrifuge tube and the tubes were centrifuged for 20 minutes at 14,000rpm at 4°C. 50ul of the supernatant was taken and labeled as sample 2. The liquid supernatant was transferred into a 15mL conical tube leavening the insoluble materials behind.
The lysate was filtered through a syringe filter with a 5 mL syringe into a 14mL round bottom transformation tube. Then, 0.5mL of Ni-NTA resin/buffer mix was added. The apparatus shown to the right was set up along with an ice bucket and column (Bio Rad Laboratories, Hercules, CA). After running nanopure water andresin/buffer through the column into the 10mL “waste” round bottom tube, 50ul was taken from the waste solution and was labeled as sample 3. The Ni-NTA resin was washed with 5ml of 20mM imidazole in 1XPBS. The solution was collected into the 10ml “wash” round bottom tube. 50ul was sampled and were labeled as sample 4.
After adding 50mL of the buffer containing 250 mM of imidazole, the buffer was collected in a 15mL conical tube labeled as “Elution 1”. Next, 5 mL was elution buffer was added again and the solution was collected in a 15mL conical tube labeled as “Elution 2”. In the end, 50ul of each elution1 and elution 2 was taken and they were labeled as sample 5 and sample 6.
By using the Nanodrop spectrophotometer (thermo Scientific, Wilmington, DE) with elution #1, the concentration of the purified protein was estimated. The absorptions were measured at both Protein A280 mode and UV/VIS mode. After searching the extinction coefficient online along with the absorbance measured, both the concentration and yield of the purified protein were measured at 280 nm and maximal wavelength.
In order to prepare the SDS-PAGE gel samples, sample 1 was centrifuged at 5 minutes at 5,000 rpm. After decanting the liquid, 200ul of water and 40ul of loading buffer was added. For samples 2-6 (4-6 from partner), 10ul of 6x loading buffer was added. All the samples were placed in a heat block at 95°C for five minutes and were later centrifuged for 2minutes at 5,000 rpm. After assembling mini-PROTEAN tank (Bio Rad Laboratories, Hercules, CA), each lane was cleaned using needle and syringe. In the first lane, 7ul of MW standard (Fermentas, Glen Burnie, MD) was added. Afterwards, 20ul of each protein samples was sequentially added (MW-1-2-3-4-5-6-[4-5-6]). The gel ran for 25 minutes at 200V. After sufficiently rinsing the gel, ~30mL of imperial protein stain was added. The container with the gel was placed on the orbital shaker for 1.5 hours. After rinsing the gel twice, KimWipe was placed in the container and the gel was washed overnight. After placing the gel on a Whatman filter paper and covering with saran wrap, the gel was dried at 75°C on gradient cycle for 1.5 hours.
Results:
Wet Pellet Weight: .42g
Most gbr22 protein contained in Elution #1 after lysing cell, centrifuging, and filtering through Ni-NTA affinity purfication
Elution #2 as backup
Absorption at 0.896 at wavelength of 280nm
Absorption at 0.869 at wavelength of 280nm
Absorption measured at 0.136 at the wavelength of 574 nm
Absorption measured at 0.126 at the wavelength of 574 nm
Beer's Law Calculation
>Determining Concentration of Protein using 280nm
Molecular weight: 25794.2 g/mol
Extinction Coefficient: 38850 M^-1*CM^-1
A=Ebc
A=(.896+.869)/2 =.8825
.8825= c (1) (38850 M^-1*CM^-1)
c=.00002273 mol/L
>Determining Concentration of Protein using Maximal Wavelength
A= (.136+.126)/2 = .131
.131 = c (1) (118,300 M^-1*CM^-1)
c= .00000111 mol/L
Yields from Nanodrop Spectrophotometry
>280nm wavelength: .00002775 mg
(.0002272mol/L)(25794.2g/mol)= .00000555 g/L
.00000555 mg/ml (5ml)= .00002775 mg
>Maximal wavelength: .057926312 mg
(.00000111mol/L)(25794.2g/mol)= .02863156 g/L
(.0286315mg/mL) (5mL) = .05792312 mg
(Left to Right: MW-1-2-3-4-5-6-[4-5-6 sample from partner])
Discussion
Since the lab outlined a detailed procedure, there could be numerous sources of error that could mislead the data. When working with E. coli and bacterial media in protein expression, aseptic technique was crucial to prevent contamination. For example, a gas burner was turned on to sterilize the general area when handling the bacteria. If the tubes were left open for a long period of time or proper sterile equipment was not used, the results would show inconsistencies. In addition, including bacteria, that are not resistant to ampicillin or contain mutation, would provide inaccurate outcome from protein purification and characterization. Lastly, the lab required using unfamiliar equipment, such as the Nanodrop spectrophotometer and mini-PROTEAN tank. Misusing the equipment could lead to incorrect reading of measurements that could significantly impact the results.
The protein purification steps successfully separated the protein from the overexpressed bacteria. By adding lysozyme and Benzonase, the soluble materials held within the cell were released. Then, the centrifugation removed the insoluble large cell materials. Using affinity tag and Ni-NTA resin, all proteins except gbr22 were filtered.
Samples 1-6, used for gel electrophoresis, represented various specimens taken during the protein expression and purification steps. The first sample taken contains the gbr22 protein, which includes both soluble and insoluble cell materials grown overnight before any filtration. Before the second sample, lysozyme was used to break down the cell wall and lyse the cell. Moreover, Benzonase digested the DNA/RNA and reduced the viscosity of the mixture. As a result, sample 2 contained everything except the large insoluble protein particles filtered by centrifugation. Sample 3 involved a simple flow, which kept all the proteins including gbr22. Before taking the fourth sample, a small amount of imidazole was added to remove the proteins that were only loosely bound to the resin. Sample 5 contained the purified gbr22 proteins detached from the nickel beads since a large amount of imidazole was added. Lastly, sample 6 contained the remaining purified proteins that were not released in sample 5. The significant changes in the amount of cellular materials in each sample can be seen in Figure 9.
Wash buffer contained less concentrated imidazole that washed away other proteins with two or three histidines while keeping the gbr22 protein with six histidines. In comparison, the elution buffer contained concentrated imidazole that had the ability to push out the gbr22 protein. These two buffers were used in the experiment since the gbr22 protein has been modified to have six histidine residues attached to the C-terminus. As a result, the histidine residues were attached to the nickel beads in the column. Adding a large amount of imidazole competes with the histidine residues for metal binding. As a result, the gbr22 protein was finally be released.
Then, the elution #1 shown in Figure 4 was used for Nanodrop spectrophotometer in order to find the absorbance at 280nm and maximal wavelength. After obtaining the extinction coefficient from external sources, the concentrations of the protein were calculated through Beer’s Law. Moreover, the amount of purified protein in mg was calculated at 280nm and maximal wavelength by knowing volume and concentration of the purified protein.
Using the dried gel and protein ladder of Molecular Weight standard in Figure 10, the purified protein was estimated to be 28 kDa. As shown in samples 2-4 in Figure 9, the presence of an extra band represented contamination. However, multiple filtrations between the samples decreased the intensity of the extra band with the progression of the lab. In the end, sample 5 contained closely purified gbr22 protein.
Conclusions
The purpose of the lab was to overexpress a recombinant protein in E. coli, to purify gbr22 protein through various filtration methods, and to analyze the samples through gel electrophoresis and Nanodrop spectrophotometer. Verified through the gel, the gbr22 protein was closely purified with little interference from extra cellular materials in sample 5. The techniques gained throughout the lab would frequently be used in independent research when a specific protein needs to be isolated to bind with a top ranking ligands from GOLD.
References
[1] Nat Methods. Protein production and purification. 2008, 5(2):135-46.
[2] European Molecule Biology Laboratory. Protein Expression and Purification Core Facility. http://www.embl.de/pepcore/pepcore_services/index.html (accessed April 14, 2012)